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Characterization of three proteins stimulating RNA polymerase II
Volume
130, number
1
FEBS LETTERS
CHARACTERIZATION Yoshinobu
NAKANISHI,
OF THREE PROTEINS
Yoshihiro
STIMULATING
RNA POLYMERASE
II
MITSUHASHI, Kazuhisa SEKIMIZU, Haruhiko YOKOI, Yasuo TANAKA, Masami HORIKOSHI and Shunji NATORI
Faculty of Pharmaceutical Sciences, Received
University of Tokyo, Hongo, Bunkyo-ku,
16 April 1981; revised version received
1. Introduction We have purified three proteins (S-II, S-II’, S-I(b)) that specifically stimulate the activity of RNA polymerase II of Ehrlich ascites tumor cells [ 1,2]. Structural studies showed that S-II’ was a phosphorylated form of S-II [3] and that radioiodinated S-I(b) and S-II’ gave identical peptide maps, whereas the M, of S-I(b) (24 000) was significantly different from that of S-II’ (41 000) [2]. These proteins were found to share common antigenicity and to be located entirely in the nucleoplasm, not in the nucleoli [4]. Antibodies raised against S-II selectively inhibited a-amanitin-sensitive RNA synthesis in isolated nuclei, indicating that these proteins are essential for transcription by RNA polymerase II in vivo [5]. Moreover, when S-II was added exogenously to isolated nuclei from spleen cells of anemic mice, it significantly enhanced the synthesis of globin mRNA as well as cw-amanitin-sensitive RNA synthesis [6]. This paper describes studies on the optimum conditions for stimulation of RNA polymerase II using purified stimulatory proteins and enzyme. Under these conditions, RNA polymerase II and a stimulatory protein are suggested to interact in a molar ratio of 1: 1, and it was possible to obtain significant stimulation with a few nanograms of purified stimulatory proteins. These proteins did not seem to participate in the initiation of RNA synthesis from the promoter region of the adenovirus major late gene, under these conditions.
2. Materials and methods RNA polymerase II was purified to homogeneity from Ehrlich ascites tumor cells by the following ElsevierlNorth-Holland
July 1981
Biomedical Press
Tokyo, 113 Japan
26 May 1981
procedures: chromatography on DEAE-cellulose, phosphocellulose, DEAE-Sephadex, sucrose density gradient centrifugation and finally a 2nd DEAESephadex chromatography (in preparation). The final preparation gave a single band by non-denaturing gel electrophoresis and it separated into 11 polypeptides on SDS-polyacrylamide gel. Stimulating proteins S-II and S-II’ were purified as in [l] and S-I(b) was purified as in [2]. The preparations each gave a single band by SDS-polyacrylamide gel electrophoresis.
3. Results We established the optimum conditions for stimulation of RNA polymerase II using the 3 purified stimulatory proteins and purified RNA polymerase II from Ehrlich ascites tumor cells. These conditions are summarized in the legend to fig.1. Under the conditions in [ 11, stimulation was <5-6-fold, whereas under these conditions it was >20-fold. The main difference between the two conditions was the salt concentration. The effect of (NH4)2S04 on the stimulation of RNA synthesis under the new conditions is shown in fig.l(A). The activity of RNA polymerase II alone increased gradually with increase in the concentration of (NH,),SO+ However, RNA synthesis in the presence of a stimulatory protein was maximum when (NH&SO4 in the reaction mixture was O-5 mM. The stimulation decreased markedly at higher concentrations of (NH&SO4 and almost no stimulation was detected at 50 mM. The effect of (NH&SO4 on stimulation of RNA synthesis was essentially the same with all 3 stimulatory proteins. Thus, it was concluded that (NH&SO4 was not necessary to measure the overall stimulatory effect of these proteins. The stimulatory effects of the 3 proteins were also 69
Volume
130. number
1
MnCI,
(mM)
FEBS LETTERS
o-0 0
July 1981
c’rii4;sch (mM)
v”O Fig.1. Effects of (NH,),SO, and MnCl, on the activity of the stimulatory proteins of RNA polymerase II: 0.4 pg RNA polymerase II was assayed in the absence (0) or presence of S-II (IO ng (o)), S-II’ (IO ng (=)) and S-I(b) (6 ng (A)), respectively, with increasing amounts of (A) (NH,),SO, and (B) MnCl,. The assay medium contained, in a total volume of 250 ~1, 15 mM of TrisHCI (pH 7.9), 2 mM p-mercaptoethanol, 2 mM MnCI,, 200 WM each of ATP, GTP and CTP, 20 PM of UTP, 1 PuCi [ ‘H]UTP (20 Ci/mmol), I .25 pg purified Ehrlich ascites tumor DNA, 13% (v/v) glycerol, 0.04% (v/v) Triton X-100 and purified RNA polymerase II. The medium was incubated for 30 min at 37°C in the presence or absence of stimulatory proteins and incorporation of UMP into the acid-insoluble fraction was measured.
greatly affected [MnClJ in the reaction mixture. As shown in f&.1(B), MnCl, was essential for the expression of stimulatory activity, having a very narrow optimum concentration range of l-2 mM; higher [MnCl,] decreased the stimulatory activity markedly. Similar results on the effect of MnCl, were obtained with all 3 stimulatory proteins. It was found that MnCl, could not be replaced by MgClz and that MgClz was not essential for either RNA polymerase II itself or stimulation of RNA synthesis (not shown). Addition of 0.04% (v/v) of Triton X-100 was effective for stabilizing the activity of RNA polymerase II. The 3 stimulatory proteins had qualitative similar stimulatory effects on overall RNA synthesis. The dosedependencies of the effects of S-II and S-I(b) were tested under the optimal conditions using 1 pg RNA polymerase II. As shown in fig.2, a few nanograms of S-II or S-I(b) markedly enhanced the activity of RNA polymerase II, which reached a plateau with -50-100 ng, The same result was obtained with S-II’. Since the fl4, of RNA polymerase II was 70
20
40
60
80
100
Amount of Factors hg) Fig.2. Dose-responses of S-I(b) (0) and S-II (0): 1 fig RNA polymerase II was assayed with increasing amounts of S-I(b) and S-II.
calculated to be 480 000, it is likely that the stimulatory activity reached a plateau when the molar ratio of RNA polymerase II to stimulatory protein(s) was -1: 1 . Thus, probably these proteins interact with RNA polymerase II stoichiometrically, not catalytically. The next question was what kind of RNA is synthesized in the presence of these proteins. To answer this question we employed a truncated DNA of adenovirus major late gene as template. It is known that this DNA contains only one initiation site for RNA polymerase II, and that when it is used as template for RNA synthesis in a HeLa cell lysate in which faithful transcription occurs [7], the major product is RNA 536 nucleotides long which starts from the correct initiation site [S]. If the stimulatory proteins are essential to allow RNA polymerase II to distinguish the correct initiation site and if the enhanced RNA synthesis starts from this site, the product should give a discrete band of RNA of 536 nucleotides on acrylamide gel. To examine this point, RNA synthesis was performed using purified RNA polymerase II in the presence or absence of stimulatory proteins, and the resulting RNA was extracted and analyzed. Incorporation of [a-32P] UMP into the acid-insoluble fraction was enhanced -lo-fold in the presence of each stimulatory protein. An autoradiography of the RNA is shown in fig.3. No discrete band was detected when RNA synthesized by purified RNA polymerase II with
Volume
130. number
1
FEBS LETTERS
2m.192
Fig.3. Analysis of RNA synthesized in vitro. RNA synthesis was performed in the presence of each stimulatory protein in reaction mixtures containing 0.5 $Zi [w~*P]UTP and 0.25 yg ~~4Idigest ofpBR313 DNA Conta~~g~~~I-F of adenovirus major late gene as in [S]. RNA was extracted and electrophoresed on 5% (w/v) acrylamide-7 M urea gel as in [ 111, then autoradiographed: (1) terminally labeled @X 174 XhaI fragments (used as size markers); (2) background (without template DNA); (3) RNA synthesized by RNA polymerase IL alone; (4-6) RNA synthesized in the presence of S-I(b), S-H and S-II’, respectively; (7) RNA synthesized in HeLa cell &sate 171 with the same template. The arrow indicates the position of RNA having 536 nucleotides.
or without stimulatory proteins was analyzed. The RNA produced gave a smear on autorad~ography and only its intensity increased on addition of a stimulatory protein. Thus random RNA synthesis occurred under these conditions and the stimulatory proteins enhanced this RNA synthesis. No significant difference was detected in the autoradiographic patterns of the products with the 3 stimulator proteins. As a control, RNA synthesized in a HeLa celt lysate is shown in lane 7. In this case a discrete band of 536 nucleotides is clearly visible.
July 1981
found that the 3 purified stimulatory proteins, which are immunologically cross-reactive, respond in the same way to changes in [MnCI,] and ](NH&S04]. The stimulator proteins were very susceptible to the presence of these two components in the reaction mixture, and their optimum conditions for stimulation of RNA synthesis were the same. Thus, we could not detect any qualitative difference between these proteins as far as overall stimulation of RNA synthesis in vitro was concerned, In addition to RNA polymerase II, some protein factor(s) is (are) essential for the initiation of RNA synthesis from the correct initiation site [8,10]. We found that the 3 stimulatory proteins did not induce correct initiation in vitro with truncated adenovirus DNAcontaining the late promotor region as template. However, we could not exclude the possibility that these stimulatory proteins may be some of several essential proteins involved in initiation of RNA synthesis from the promoter region. Since these proteins were shown to participate in meaningful RNA synthesis in isolated nuclei [S ,6],we assume that they are modulators of the rate of RNA synthesis in vivo by interacting with RNA polymerase II. The rate of RNA synthesis may differ for various genes and thus its control should be important in the regulation of eukaryotic gene expression.
Acknowledgements We are grateful to Dr Y. Suzuki for providing truncated adenovirus major late gene containing the promoter region and HeLa cell lysate for faithful transcription. This study was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References 4. Discussion There are several reports on the multiplicity of stimulatory proteins of RNA polymerase II [9]. This multiplicity is partly due to modification of a single protein. Then the question arose of whether there was any functioxlal difference in the modified proteins. We
11] Sekimizu, K., Nakanishi,
Y., Mizuno, D. (1979) Biochemistry 18,1582-1588. [2] Nakanishi, Y., Sekimizu, K., Tamura, H. (1981) submitted. [ 3) Sekimizu, K., Kubo, Y ., Segawa, K. and (1981) Biochemistry 20,2286-2292. [4] Sekimizu,K.,Mizuno, D. and Natofi, S. Ceil Res. 124,63372.
and Natori,
S.
and Natori,
S.
Natori,
S.
(1979)
Exp.
71
Volume
130, number
1
FEBS LETTERS
[5] Ueno, K., Sekimizu, K., Mizuno, D. and Natori, S. (1979) Nature 277,145-146. [6] Ueno, K., Sekimizu, K., Obinata, M., Mizuno, D. and Natori, S. (1981) Biochemistry 20,634-640. [7] Manley,J.L.,Fire,A.,Cano,A.,Sharp,P.A.and Gefter, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3855-3859. [8] Luse,D.S.andRoeder,R.G.(1980)CelJ20,691-699.
72
July 1981
[9] Wang, T. Y. and Kostraba, N. C. (1978) The Cell Nucleus, vol. 4, Chromatin, pt A (Busch, J. ed.) pp. 289-317, Academic Press, New York. [lo] Wasylyk, B., Kedinger, C., Corden, J., Brison, 0. and Chambon, P. (1980) Nature 285,367-373. [II] Maxam,A.M.andGilbert,W.(1980)MethodsEnzymol. 65/I, 4999560.
130, number
1
FEBS LETTERS
CHARACTERIZATION Yoshinobu
NAKANISHI,
OF THREE PROTEINS
Yoshihiro
STIMULATING
RNA POLYMERASE
II
MITSUHASHI, Kazuhisa SEKIMIZU, Haruhiko YOKOI, Yasuo TANAKA, Masami HORIKOSHI and Shunji NATORI
Faculty of Pharmaceutical Sciences, Received
University of Tokyo, Hongo, Bunkyo-ku,
16 April 1981; revised version received
1. Introduction We have purified three proteins (S-II, S-II’, S-I(b)) that specifically stimulate the activity of RNA polymerase II of Ehrlich ascites tumor cells [ 1,2]. Structural studies showed that S-II’ was a phosphorylated form of S-II [3] and that radioiodinated S-I(b) and S-II’ gave identical peptide maps, whereas the M, of S-I(b) (24 000) was significantly different from that of S-II’ (41 000) [2]. These proteins were found to share common antigenicity and to be located entirely in the nucleoplasm, not in the nucleoli [4]. Antibodies raised against S-II selectively inhibited a-amanitin-sensitive RNA synthesis in isolated nuclei, indicating that these proteins are essential for transcription by RNA polymerase II in vivo [5]. Moreover, when S-II was added exogenously to isolated nuclei from spleen cells of anemic mice, it significantly enhanced the synthesis of globin mRNA as well as cw-amanitin-sensitive RNA synthesis [6]. This paper describes studies on the optimum conditions for stimulation of RNA polymerase II using purified stimulatory proteins and enzyme. Under these conditions, RNA polymerase II and a stimulatory protein are suggested to interact in a molar ratio of 1: 1, and it was possible to obtain significant stimulation with a few nanograms of purified stimulatory proteins. These proteins did not seem to participate in the initiation of RNA synthesis from the promoter region of the adenovirus major late gene, under these conditions.
2. Materials and methods RNA polymerase II was purified to homogeneity from Ehrlich ascites tumor cells by the following ElsevierlNorth-Holland
July 1981
Biomedical Press
Tokyo, 113 Japan
26 May 1981
procedures: chromatography on DEAE-cellulose, phosphocellulose, DEAE-Sephadex, sucrose density gradient centrifugation and finally a 2nd DEAESephadex chromatography (in preparation). The final preparation gave a single band by non-denaturing gel electrophoresis and it separated into 11 polypeptides on SDS-polyacrylamide gel. Stimulating proteins S-II and S-II’ were purified as in [l] and S-I(b) was purified as in [2]. The preparations each gave a single band by SDS-polyacrylamide gel electrophoresis.
3. Results We established the optimum conditions for stimulation of RNA polymerase II using the 3 purified stimulatory proteins and purified RNA polymerase II from Ehrlich ascites tumor cells. These conditions are summarized in the legend to fig.1. Under the conditions in [ 11, stimulation was <5-6-fold, whereas under these conditions it was >20-fold. The main difference between the two conditions was the salt concentration. The effect of (NH4)2S04 on the stimulation of RNA synthesis under the new conditions is shown in fig.l(A). The activity of RNA polymerase II alone increased gradually with increase in the concentration of (NH,),SO+ However, RNA synthesis in the presence of a stimulatory protein was maximum when (NH&SO4 in the reaction mixture was O-5 mM. The stimulation decreased markedly at higher concentrations of (NH&SO4 and almost no stimulation was detected at 50 mM. The effect of (NH&SO4 on stimulation of RNA synthesis was essentially the same with all 3 stimulatory proteins. Thus, it was concluded that (NH&SO4 was not necessary to measure the overall stimulatory effect of these proteins. The stimulatory effects of the 3 proteins were also 69
Volume
130. number
1
MnCI,
(mM)
FEBS LETTERS
o-0 0
July 1981
c’rii4;sch (mM)
v”O Fig.1. Effects of (NH,),SO, and MnCl, on the activity of the stimulatory proteins of RNA polymerase II: 0.4 pg RNA polymerase II was assayed in the absence (0) or presence of S-II (IO ng (o)), S-II’ (IO ng (=)) and S-I(b) (6 ng (A)), respectively, with increasing amounts of (A) (NH,),SO, and (B) MnCl,. The assay medium contained, in a total volume of 250 ~1, 15 mM of TrisHCI (pH 7.9), 2 mM p-mercaptoethanol, 2 mM MnCI,, 200 WM each of ATP, GTP and CTP, 20 PM of UTP, 1 PuCi [ ‘H]UTP (20 Ci/mmol), I .25 pg purified Ehrlich ascites tumor DNA, 13% (v/v) glycerol, 0.04% (v/v) Triton X-100 and purified RNA polymerase II. The medium was incubated for 30 min at 37°C in the presence or absence of stimulatory proteins and incorporation of UMP into the acid-insoluble fraction was measured.
greatly affected [MnClJ in the reaction mixture. As shown in f&.1(B), MnCl, was essential for the expression of stimulatory activity, having a very narrow optimum concentration range of l-2 mM; higher [MnCl,] decreased the stimulatory activity markedly. Similar results on the effect of MnCl, were obtained with all 3 stimulatory proteins. It was found that MnCl, could not be replaced by MgClz and that MgClz was not essential for either RNA polymerase II itself or stimulation of RNA synthesis (not shown). Addition of 0.04% (v/v) of Triton X-100 was effective for stabilizing the activity of RNA polymerase II. The 3 stimulatory proteins had qualitative similar stimulatory effects on overall RNA synthesis. The dosedependencies of the effects of S-II and S-I(b) were tested under the optimal conditions using 1 pg RNA polymerase II. As shown in fig.2, a few nanograms of S-II or S-I(b) markedly enhanced the activity of RNA polymerase II, which reached a plateau with -50-100 ng, The same result was obtained with S-II’. Since the fl4, of RNA polymerase II was 70
20
40
60
80
100
Amount of Factors hg) Fig.2. Dose-responses of S-I(b) (0) and S-II (0): 1 fig RNA polymerase II was assayed with increasing amounts of S-I(b) and S-II.
calculated to be 480 000, it is likely that the stimulatory activity reached a plateau when the molar ratio of RNA polymerase II to stimulatory protein(s) was -1: 1 . Thus, probably these proteins interact with RNA polymerase II stoichiometrically, not catalytically. The next question was what kind of RNA is synthesized in the presence of these proteins. To answer this question we employed a truncated DNA of adenovirus major late gene as template. It is known that this DNA contains only one initiation site for RNA polymerase II, and that when it is used as template for RNA synthesis in a HeLa cell lysate in which faithful transcription occurs [7], the major product is RNA 536 nucleotides long which starts from the correct initiation site [S]. If the stimulatory proteins are essential to allow RNA polymerase II to distinguish the correct initiation site and if the enhanced RNA synthesis starts from this site, the product should give a discrete band of RNA of 536 nucleotides on acrylamide gel. To examine this point, RNA synthesis was performed using purified RNA polymerase II in the presence or absence of stimulatory proteins, and the resulting RNA was extracted and analyzed. Incorporation of [a-32P] UMP into the acid-insoluble fraction was enhanced -lo-fold in the presence of each stimulatory protein. An autoradiography of the RNA is shown in fig.3. No discrete band was detected when RNA synthesized by purified RNA polymerase II with
Volume
130. number
1
FEBS LETTERS
2m.192
Fig.3. Analysis of RNA synthesized in vitro. RNA synthesis was performed in the presence of each stimulatory protein in reaction mixtures containing 0.5 $Zi [w~*P]UTP and 0.25 yg ~~4Idigest ofpBR313 DNA Conta~~g~~~I-F of adenovirus major late gene as in [S]. RNA was extracted and electrophoresed on 5% (w/v) acrylamide-7 M urea gel as in [ 111, then autoradiographed: (1) terminally labeled @X 174 XhaI fragments (used as size markers); (2) background (without template DNA); (3) RNA synthesized by RNA polymerase IL alone; (4-6) RNA synthesized in the presence of S-I(b), S-H and S-II’, respectively; (7) RNA synthesized in HeLa cell &sate 171 with the same template. The arrow indicates the position of RNA having 536 nucleotides.
or without stimulatory proteins was analyzed. The RNA produced gave a smear on autorad~ography and only its intensity increased on addition of a stimulatory protein. Thus random RNA synthesis occurred under these conditions and the stimulatory proteins enhanced this RNA synthesis. No significant difference was detected in the autoradiographic patterns of the products with the 3 stimulator proteins. As a control, RNA synthesized in a HeLa celt lysate is shown in lane 7. In this case a discrete band of 536 nucleotides is clearly visible.
July 1981
found that the 3 purified stimulatory proteins, which are immunologically cross-reactive, respond in the same way to changes in [MnCI,] and ](NH&S04]. The stimulator proteins were very susceptible to the presence of these two components in the reaction mixture, and their optimum conditions for stimulation of RNA synthesis were the same. Thus, we could not detect any qualitative difference between these proteins as far as overall stimulation of RNA synthesis in vitro was concerned, In addition to RNA polymerase II, some protein factor(s) is (are) essential for the initiation of RNA synthesis from the correct initiation site [8,10]. We found that the 3 stimulatory proteins did not induce correct initiation in vitro with truncated adenovirus DNAcontaining the late promotor region as template. However, we could not exclude the possibility that these stimulatory proteins may be some of several essential proteins involved in initiation of RNA synthesis from the promoter region. Since these proteins were shown to participate in meaningful RNA synthesis in isolated nuclei [S ,6],we assume that they are modulators of the rate of RNA synthesis in vivo by interacting with RNA polymerase II. The rate of RNA synthesis may differ for various genes and thus its control should be important in the regulation of eukaryotic gene expression.
Acknowledgements We are grateful to Dr Y. Suzuki for providing truncated adenovirus major late gene containing the promoter region and HeLa cell lysate for faithful transcription. This study was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References 4. Discussion There are several reports on the multiplicity of stimulatory proteins of RNA polymerase II [9]. This multiplicity is partly due to modification of a single protein. Then the question arose of whether there was any functioxlal difference in the modified proteins. We
11] Sekimizu, K., Nakanishi,
Y., Mizuno, D. (1979) Biochemistry 18,1582-1588. [2] Nakanishi, Y., Sekimizu, K., Tamura, H. (1981) submitted. [ 3) Sekimizu, K., Kubo, Y ., Segawa, K. and (1981) Biochemistry 20,2286-2292. [4] Sekimizu,K.,Mizuno, D. and Natofi, S. Ceil Res. 124,63372.
and Natori,
S.
and Natori,
S.
Natori,
S.
(1979)
Exp.
71
Volume
130, number
1
FEBS LETTERS
[5] Ueno, K., Sekimizu, K., Mizuno, D. and Natori, S. (1979) Nature 277,145-146. [6] Ueno, K., Sekimizu, K., Obinata, M., Mizuno, D. and Natori, S. (1981) Biochemistry 20,634-640. [7] Manley,J.L.,Fire,A.,Cano,A.,Sharp,P.A.and Gefter, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3855-3859. [8] Luse,D.S.andRoeder,R.G.(1980)CelJ20,691-699.
72
July 1981
[9] Wang, T. Y. and Kostraba, N. C. (1978) The Cell Nucleus, vol. 4, Chromatin, pt A (Busch, J. ed.) pp. 289-317, Academic Press, New York. [lo] Wasylyk, B., Kedinger, C., Corden, J., Brison, 0. and Chambon, P. (1980) Nature 285,367-373. [II] Maxam,A.M.andGilbert,W.(1980)MethodsEnzymol. 65/I, 4999560.